Attachment of M. pneumoniae to many cell types in vitro has been shown to be mediated by host cell surface molecules containing terminal sialic acid moieties. We have previously shown that M. pneumoniae binds strongly to glycolipids with terminal sulfated galactose units, but only weakly to common glycolipids containing the requisite terminal sialyllactosamine structures. The pathogen binds very strongly, however, to glycoproteins possessing terminal sialyllactosamine with the a2-3 linkage, but not to glycoproteins with the a2-6 linkage. Affinity columns of dextran sulfate and fetuin have been employed to isolate M. pneumoniae proteins that specifically bind to the two classes of tissue receptors. A previously described 32 kDa protein, as well as a 56 kDa protein were among those binding avidly to the dextran sulfate column. Whole cell iodination, surface proteolysis, and membrane preparations were used to determine that these proteins are surface exposed. Monospecific sera to the 32 kDa protein and a monoclonal antibody to the 56 kDa protein could each be used to inhibit binding of M. pneumoniae to MRC-5 cell cultures. These data suggest that at least two proteins, in addition to the well-characterized adhesin P1, are involved in attachment of M. pneumoniae to receptors on host tissues. These studies have provided information concerning mechanisms by which Mycoplasma pneumoniae can establish infection via attachment. In addition, the information is relevant to our attempts to develop effective vaccines for the prevention of the primary atypical pneumonia which is caused by this pathogen.